Glutamine is an essential nutrient for the growth and maintenance of mammalian cells in culture, and tumor cells are avid glutamine consumers. However, little is known about the mechanism by which glutamine supports tumor cell proliferation and viability. This revised application will test the overall hypothesis that glutamine provides tumor cells with reductive equivalents as well as precursors for glutathione synthesis, a molecule which is essential for cellular protection against chemical and oxidative damage. Glutamine utilization may contribute to cellular redox metabolism and the mechanism by which cells respond to oxidative and xenobiotic stress. A series of human breast cell lines exhibiting a wide range of glutamine dependence for growth and viability, as well as a wide range of glutamine utilization rates, have been identified. In addition, the response of two highly glutamine-sensitive breast cell lines to glutamine starvation has been characterized and will be used to directly test a number of hypotheses: 1) Glutamine is-used to provide glutamate and cystine for synthesis of glutathione. 2) Glutamine utilization provides reductive equivalents to tumor cell mitochondria and therefore controls tumor cell growth and viability through mitochondrial redox mechanisms. 3) Targeted inhibition of glutaminase expression, the first enzyme involved in glutaminolysis, represses glutamine utilization and thereby inhibits growth and increases the sensitivity of tumor cells to chemotherapeutic drugs which are detoxified by glutathione. In general, methods to block the use of glutamine by tumor cells will be developed, and the efficacy of this strategy for inhibiting tumor growth or for increasing the sensitivity of tumors to chemotoxic drugs will be examined. This may provide the basis for novel metabolically directed cancer therapies.